NK cells (or "Natural Killer" cells) are lymphoctyes found in the peripheral blood, in bone marrow and in other lymphoid tissues. NK cells are not considered to be T cells because they do not rearrange or productively transcribe the .alpha., B, .gamma. or .delta. genes of the T cell antigen receptor complex (or "TcR"). NK cells have ability to kill and lyse certain tumor and virally infected cells. Unlike T cells, however, the killing activity of these cells is not restricted by the major histocompatiblity complex (or "MHC"). NK cells also secrete interferons and cytokines that regulate immune responses and hematopoiesis.
Morphologically, NK cells usually are large granular lymphocytes with prominent azurophilic granules. NK cells also may be identified by the expression of certain markers or structures on their cell surface. These structures can be detected by monoclonal antibodies which have been prepared by conventional methods against them.
The prototypic structures (or "antigens") expressed by the majority of NK cells are CD16 and CD56. (The CD or "Cluster Designation" is an international standard used in the field to define a particular antigen by its molecular weight, cell type distribution and other criteria. The most recent Workshop to cluster antigens and thus antibodies thereto was held in Vienna in February 1989. The most recent list of cluster designations has been published in Journal of Immunology, 143:758 (1989)).
CD16 is an antigen of approximately 50-70 kD and is an Fc receptor for IgG. Besides NK cells, CD16 is expressed on granulocytes, activated macrophages and a minor subset of T cells. Functionally, the binding of immune complexes (e.g. antibody coated target cells) or anti-CD16 monoclonal antibodies to CD16 on NK cells causes a rapid increase in intracellular Ca.sup.++ and inositol trisphosphate (or "IP.sub.3 ") generation with subsequent transcription of lymphokines and triggering of cell mediated toxicity.
Two highly homologous genes encode CD16. CD16-I encodes a phosphatidylinositol(PI) glycan linked glycoprotein expressed by granulocytes, whereas CD16-II encodes a transmembrane-anchored glycoprotein expressed on NK cells and on activated macrophages. The expression of CD16 II on the surface of COS-7 cells (a monkey kidney tumor cell line, ATCC CRL 1651) requires co-transfection with the .gamma. subunit of the high affinity IgE receptor or the CD3.zeta. chain.
Monoclonal antibodies can be raised against CD16 by conventional techniques such as those described by Kohler and Milstein. See Nature, 256:495 (1975). For example, mice can be immunized with large granular lymphocytes (or NK cells) isolated from human peripheral blood. Spleen cells from mice so immunized then can be hybridized to a mouse plasmacytoma cell line, such as P3-X63 (ATCC TIB 9), in the presence of a agent such as polyethylene glycol. Clones then can be screened for reactivity with NK cells. Anti CD16 monoclonal antibodies are commercially available from Becton Dickinson Immunocytometry Systems (or "BDIS") as Anti-Leu 11.
CD56 is an antigen of approximately 145-220 kD. It is expressed on essentially all resting and activated NK cells, on approximately 5% of T cells but not on granulocytes, monocytes or B cells. Anti-CD56 monoclonal antibodies can be prepared as above using the KG-1a cell line (a human hematopoietic cell line, ATCC CCL 246.1) as an immunogen. Commercially, an anti-CD56 monoclonal antibody is available from BDIS as Anti-Leu 19.
Not all NK cells, however, express CD16. Similarly, there are NK cells that express quantitatively less CD56. Using anti-CD16 and anti-CD56 monoclonal antibodies which have been fluorescently labelled by conventional techniques with chromophores such as phycoerythrin and fluorescein isothiocyanate in combination with a flow cytometer, three distinct subsets of NK cells are found: 1) a small population of CD56.sup.bright, CD16.sup.negative cells; 2) a small population of CD56.sup.bright, CD16.sup.dim cells; and 3) a major population of CD56.sup.dim, CD16.sup.bright cells ("Bright" and "dim" refer to the fluorescence intensity of the cell stained with a labelled monoclonal antibody. The cells that express the most antigen will be "bright", those that express less antigen will be "dim" and those that express no antigen will be "negative.") These populations are referred to as CD16.sup.-, CD16hu + and CD16.sup.++ NK cells, respectively. The CD16.sup.++ population comprises .about.15% of total peripheral blood lymphoctyes (or "PBL") while the CD16.sup.+ and CD16.sup.- populations comprise less than 1% of PBL. It is thought that CD16.sup.+ cells eventually become CD16.sup.++.
Besides expressing CD16 and CD56, NK cells also express a variety of other antigens including CD2, CD7, CD11b. CD38, CD45R, CD18 and the p75 subunit of the interleukin 2 receptor (or "IL 2R"). A minor population of NK cells also express low surface density CD8. None of the CD16.sup.-, CD16.sup.+ or CD16.sup.++ NK cell populations express on their cell surface CD3 .gamma., .delta. or .epsilon. or react with antibodies that preferentially bind the .alpha./B or .gamma./.delta. subunits of TcR. Additionally, none of the populations react with antibodies against T cell associated antigens CD4, CD5 or CD28, the B cell associated antigen CD19 or the monocyte associated antigen CD14.
There are, however, differences between the three populations of NK cells. For example, 50-60% of the CD16.sup.++ NK cells express CD57, but neither the CD16.sup.+ nor the CD16.sup.- NK cell populations generally express CD57. On the other hand, both the CD16.sup.- and CD16.sup.+ populations express a high surface density of Leu-8 (a 70-80 kD antigen that the BDIS monoclonal antibody Anti Leu-8 reacts with on the majority of PBL, thymocytes and B cells and which was prepared using peripheral blood T cells as an immunogen) but only 20% of CD16.sup.++ NK cells also express Leu-8. Further, CD16.sup.++ NK cells express no CD25 but the CD16.sup.+ and CD16.sup.- NK cell populations express detectable levels of CD25.
Functionally, CD16.sup.- NK cells isolated directly from blood have little cytolytic activity against human tumor cell targets such as K-562 (ATCC CCL 243). CD16.sup.+ and CD16.sup.++ NK cells both show cytolytic activity against NK sensitive tumor cell targets with the CD16.sup.++ NK cells showing a greater activity. On the other hand, CD16.sup.- and CD16.sup.+ NK cells have a greater proliferative response to IL-2 than do CD16.sup.++ cells.
Taken together, the several NK cell populations appear to comprise a continuum of differentiation in the peripheral blood with the CD16.sup.- population being the most immature and the CD16.sup.++ population being the most mature. Accordingly, in adoptive immunotherapy, such as that described in U.S. Pat. No. 4,607,007, it may be more appropriate to use CD16.sup.- or CD16.sup.+ NK cells to achieve a greater effect.
Apart from this characterization of NK cells, other work has been done to determine how NK cells recognize a target cell in the absence of the MHC. As previously described, T cells can function in a cytolytic manner but usually do so only in the context of the MHC. It is believed that the recognition function by the T cell is mediated through the TcR but that signal transduction to turn on the cells cytolytic function occurs through the co associated CD3 structure via the PI pathway.
The T cell antigen receptor is composed of an .alpha./B or .gamma./.delta. TcR heterodimer that is non-covalently associated with CD3. CD3 is a complex of at least five subunits, designated CD3.gamma., .delta., .epsilon., .zeta. and .eta.. CD3.gamma., .delta., .epsilon., .zeta. are monomeric proteins encoded by genes closely-linked on chromosome 11, whereas CD3.zeta. is on chromosome 1 and expressed as a disulfide linked homodimer or a heterodimer in association with CD3.eta.. Antigen induced T cell activation results in tyrosine phosphorylation of CD3.zeta.. It is believed that efficient signal transduction via the CD3/TcR complex requires expression of CD3.zeta..
Because NK cells lack surface CD3 .gamma., .delta. and .epsilon. and TcR, there has been interest in determining how recognition occurs and how signal transduction occurs. Recently, it has been reported by Anderson et al., Nature, 341:159 (1989), that CD3.zeta. is expressed on NK cells. Anderson et al. further reported that there were apparently novel structures associated with CD3.zeta.. In FIG. 4, they show 12 kD, 60-70 kD and 80-90 kD proteins all associated with CD3.zeta.. Surprisingly, however, it has been discovered that the 60-70 kD structure is not novel but is, in fact, CD16. Thus, there is little information to suggest through what mechanism signal transduction occurs in NK cells that lack CD16.